pET28a(+)-SpdCas9-LBiT_Strep-Tag-II
(Plasmid #198751)

• Purpose: Expresses S. Pyogenes dCas9 fused to LargeBiT fragment of split NanoLuc luciferase
• Depositing Lab: Maarten Merkx
• Publication: van der Veer et al ACS Cent Sci. 2023 Mar 15;9(4):657-667. doi: 10.1021/acscentsci.2c01467. eCollection 2023 Apr 26.

Backbone

• Vector backbone: pET-28a(+)
• Backbone manufacturer: EMD Millipore
• Backbone size w/o insert (bp): 5369
• Total vector size (bp): 9910
• Modifications to backbone: Removed N-terminal His-tag
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: SpdCas9 - LargeBiT
• Alt name: Catalytically dead S. pyogenes Cas9 fused to LargeBiT (NanoBiT / split NanoLuc)
• Species: Synthetic; Streptococcus pyogenes
• Insert Size (bp): 4677
• Mutation: D10A, H840A (nuclease inactivating mutations in 'd' variant of SpCas9)
• Promoter: T7
• Tag / Fusion Protein:
  • Strep-tag II (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NcoI (not destroyed)
• 3′ cloning site: XhoI (not destroyed)
• 5′ sequencing primer: T7 promoter
• 3′ sequencing primer: T7 terminator

Resource Information

• Supplemental Documents:
  • pET28a(+)-SpdCas9-LBiT.gb
• A portion of this plasmid was derived from a plasmid made by: S. pyogenes dCas9 coding sequence was copied from the pET-dCas9-VP64-6xHis plasmid gifted by David Liu (Addgene plasmid #62935)

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry